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RNAi Delivery Proves Vexing

Emerging Field Simultaneously Holds Both Promise and Problems

Advances in miRNA

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The regulation of miR-211 in melanomas

Melanoma is associated with abnormal patterns of microRNA (miRNA) and large noncoding RNA (lncRNA) gene expression. Ranjan Perera, Ph.D., associate professor at Sanford-Burnham Medical Research Institute, is deciphering how miRNAs and lncRNAs impact development of this disease.

“As master regulators of hundreds of RNAs, miRNAs have been implicated in a number of malignancies. Because they may be either upregulated or downregulated, we are examining the possibility of using miRNA and lncRNAs as biomarker and discovery tools. As biomarkers, they may be useful for early prognostics or diagnostics. As discovery tools, they could be employed as therapeutics targeting certain genes or even to assist with gene-function discovery.”

Dr. Perera first looked at the miRNA signatures between normal and malignant melanocytes and melanoma cell lines. “Our gene-expression studies identified a handful of miRNAs that were downregulated. One of these is miR-211. While normal cells expressed miRNA-211, malignant cells did not. Thus, our basic hypothesis is that miRNA-211 downregulation in melanocytes induces human melanoma.”

By putting miR-211 back into melanoma cells, Dr. Perera and his team found that it blocked tumor growth and invasion. To begin to dissect the mechanism, he performed functional studies. “We found that miR-211 targets the KCNMA1 gene that codes for the so-called potassium large conductance calcium-activated channel, subfamily M, alpha member 1,” he said.

“Next, we found that miR-211 resides in the intron of TRPM1, which encodes a calcium-channel protein called the transient receptor potential cation channel subfamily M member 1 protein. The expression of TRPM1 is inversely correlated with melanoma aggressiveness, suggesting that it suppresses the metastasis of melanoma cells.”

Finally, Dr. Perera’s team found that the expression of miR-211 is regulated via TRPM1 gene that is regulated by MITF, which encodes the microphthalmia-associated transcription factor. He summarized his findings: “When we utilized siRNAs to knockdown the expression of MITF, we found that both TRPM1 and miR-211 were also knocked down.”

“We are now able to build a new signal-transduction pathway that may ultimately provide information for early melanoma prognostics,” Dr. Perera explained. “My team and others are evaluating patient samples and have already demonstrated that indeed miR-211 is associated with human melanomas.”

Cell Fate Architecture

The earliest events in human embryonic development are orchestrated by a complex, yet poorly understood, regulatory architecture that involves miRNAs. Previous work from the group of Paul Robson, Ph.D., group leader in stem cell and developmental biology at Genome Institute of Singapore, applied advanced single-cell gene-expression analysis to the developing mouse embryo.

“This clearly indicated that removal of particular transcripts was a key event in cell fate-decisions. The primary candidate to control such a mechanism is microRNAs,” he said.

Now Dr. Robson’s group is seeking to unravel these early events in human development utilizing a human ES cell (hESC) model. “We employed next-generation transcriptome analysis methods to characterize the dynamics of gene expression upon hESC differentiation.”

Dr. Robson focused on examining the differentiation of cells of trophoblast lineage. Trophoblasts are the first cells of the fertilized egg to differentiate. They constitute the outermost layer of the embryo, assist in uterine implantation, and develop into the fetal component of the placenta.

Dr. Robson’s team profiled, in tandem, polyadenylated and small RNA transcriptomes using strand-specific RNA-sequencing.

“There were several surprises in our study,” he stated. “In addition to identifying the expression of many known microRNAs not previously described in the cells of trophoblast lineage, we also saw the expression of many novel microRNAs. We found that many well-known genes are poorly annotated in their 3´ untranslated regions and are much longer than anticipated, thus greatly expanding the potential target sites of microRNAs.”

These studies have clinical potential relevance, according to Dr. Robson. “The application of next-generation sequencing technology will likely identify many new microRNAs in the human genome, some of which may be associated with diseases of placental lineage. This coupled with new studies that fetal-derived microRNAs transfer into the maternal circulation may allow fetal-derived microRNAs to serve as biomarkers in mother’s blood that could be used to noninvasively test for the early indications of diseases such as pre-eclampsia and intra-uterine growth restriction.”

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